Liquid chromatography (LC) is a technique for performing an analytical or preparative separation of a liquid-phase sample material of interest (e.g., a mixture of different chemical compounds) into constituent components. During the course of a chromatographic separation, the sample material is transported in a mobile phase (typically one or more solvents). The sample (the sample material and mobile phase in which it is dissolved) is forced through a stationary phase that is immiscible with the mobile phase. Typically, the stationary phase is provided in the form of a mass of particles (a packing or bed) supported in a column or cartridge through which the sample flows. The column bed is typically retained at each end of the column by a frit or filter that allows the sample to flow through while preventing the packing material from escaping the column. The respective compositions of the mobile phase and stationary phase are selected to cause differing components of the sample material in the column to become distributed between the mobile phase and stationary phase to varying degrees dependent on the respective chemistries of the sample material's components. Components that are strongly retained by the stationary phase travel slowly with the mobile phase, while components that are weakly retained by the stationary phase travel more rapidly. As a result, components of differing compositions become separated from each other as the mobile phase flows through the column.
In analytical separation, the components are separated to facilitate their analysis by detection and data acquisition techniques. Analytical separation typically entails the use of a small amount of material and small inside-diameter columns (e.g, less than 1 inch). In preparative separation, the components are separated to purify or isolate one or more chemical components from the starting material, which may be done for a further use such as reaction, synthesis, etc. Preparative separation may be performed on a small scale comparative to analytical separation, or may be performed on a much larger scale to purify a large quantity of sample material, and thus may utilize larger inside-diameter columns (e.g., 1-24 inches).
Typically, a sample to be separated is introduced into a column by first injecting the sample into a solvent flow stream at a point upstream of the column. The solvent flow stream may be a mixed flow stream formed by mixing the flows of a weak solvent and a strong solvent (or “modifier” solvent) in a solvent mixer upstream of the column. In this case, the sample may be injected into the mixed flow stream, i.e., downstream from the mixer. Typically, the sample is injected into the column with the strong solvent still being at a high concentration, which may cause chromatographic band broadening relative to the amount of solvent injected with the sample, and thus poor resolution, especially in preparative chromatography where larger quantities of sample are employed. The addition of a relatively weak solvent may be done to mitigate the effects of the strong solvent, but can cause the sample to crystallize in the sample loop or other tubing and clog the system.
Alternatively, the sample may be injected into the flow path of the strong solvent before the strong solvent is merged with the weak solvent in the mixer. In this case, the sample flows through the mixer before flowing into the column. This is disadvantageous because the sample may be retained by the mixer, thus contaminating the mixer and causing unwanted sample dispersion upstream of the column. Moreover, avoiding sample retention by the mixer means limiting the choice of materials utilized to construct the mixer. Additionally, in this case the strong solvent (usually methanol or acetonitrile) serves as the injection solvent for the sample. Many samples, however, are dissolved in other solvents such as dimethyl sulfoxide (DMSO), which might make a better injection solvent in certain applications. Moreover, DMSO and other sample solvents are often incompatible with the materials constituting the mixer's structure. Also, by utilizing the strong solvent as the injection solvent, the rate at which the sample is injected into the column is limited by the practical flow rates of the pump employed for flowing the strong solvent.
Another problem with known techniques for sample injection in high-pressure systems is that the sample is compressed in the sample loop or tubing due to operation of the pump. This compression can cause a pressure shock that disturbs the column bed, which may produce undesirable voids in the bed and loosen stationary phase material.
In addition, the use of a separate sample injection pump can cause carryover without much rinsing, because samples will diffuse into the sample push solvent and the walls of the injector are not sufficiently cleaned by injection solvent alone. The volume of the sample injection pump adds compliance to the system and increases the pressure shock that occurs when the sample injection pump is switched in-line with a high-pressure flow stream.
Therefore, there is a need for injecting samples into an LC column while minimizing sample dispersion upstream of the LC column. There is also a need for bypassing the solvent mixer to prevent mixer contamination with the sample or the solvent in which the sample is dissolved.